Enzyme with protease activity

ABSTRACT

An isolated and purified enzyme exhibiting protease activity at a pH of 4-7 which exhibits protease in 5% hydrogen peroxide and which is encoded by a DNA sequence which hybridizes to a DNA sequence depicted in SEQ ID NO: 1 or 2. Methods are described for using the protease compositions in reducing vescosity, cleaning contact lenses, baking, and preparing animal feed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/190,982,filed Nov. 12, 1998, now U.S. Pat. No. 5,998,190, which is acontinuation of No. 08/578,551 filed on Feb. 1, 1996, now U.S. Pat. No.5,854,050, which is a continuation of application Ser. No.PCT/DK94/00274 filed on Jul. 5, 1994, and claims priority under 35U.S.C. 119 of Danish application serial no. 0811/93 filed on Jul. 6,1993, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a DNA construct encoding an enzyme withprotease activity, a method of producing the enzyme, an enzyme withprotease activity, and an enzyme preparation containing the enzyme.

BACKGROUND OF THE INVENTION

Proteases are enzymes capable of cleaving peptide bonds. Acid proteases(i.e. proteases having an acidic pH optimum) have been found to beproduced by a number of different organism including mammals andmicrobes. For instance, microbial acid proteases have been found to beproduced by bacterial strains such as strains of Bacillus sp. (JP01240184), fungal strains, e.g. of Rizopus sp. (EP 72978), Schytalidiumsp. (JP 48091273), Sulpholobus sp. and Thermoplasma sp. (WO90 10072) andAspergillus sp. (JP 50121486, EP 82 395).

JP 3058794 discloses the cloning of a gene encoding an acid proteasefrom R. niveus and the recombinant expression thereof. The cloning andexpression of a gene from Cryphonectira parasitica encoding an asparticprotease is described by Choi et al. (1993). Takahashi et al. (1991),Inoue et al. (1991), and JP 407 5586 discloses the cloning of a genefrom Aspergillus niger encoding an acid proteinase (Protease A).

Berka et al. (1990) disclose a gene encoding the aspartic proteinaseaspergillopepsin A from Aspergillus awamori. The cloning of a geneencoding the aspartic proteinase aspergillopepsin O from Aspergillusoryzae is described by Berka et al. (1993). The cloning of a geneencoding the acid protease (PEPA) from Aspergillus oryzae is disclosedby Gomi et al. (1993).

Acid proteases are widely used industrially, e.g. in the preparation offood and feed, in the leather industry (e.g. to dehair hides), in theproduction of protein hydrolysates and in the wine making and brewingindustry.

There is a need for single-component acid proteases for many differentapplications, especially in the food and feed industry.

SUMMARY OF THE INVENTION

It is an object of the present invention to prepare a single-componentprotease.

Accordingly, in a first aspect the invention relates to a DNA constructcomprising a DNA sequence encoding an enzyme exhibiting proteaseactivity, which DNA sequence comprises the DNA sequence shown in SEQ IDNo. 1 or an analogous sequence thereof being at least 80% homologous tothe DNA sequence shown in SEQ ID No. 1.

In a second aspect the invention relates to a DNA construct comprising aDNA sequence encoding an enzyme exhibiting protease activity, which DNAsequence comprises the DNA sequence shown in SEQ ID No. 2 or ananalogous sequence thereof being at least 80% homologous to the DNAsequence shown in SEQ ID No. 2.

The DNA sequence shown in SEQ ID No. 1 encodes an enzyme which in thefollowing disclosure is referred to as Protease I. The enzyme encoded bythe DNA sequence shown in SEQ ID No. 2 is referred to as Protease II.

By a data base homology search it has been found that the DNA sequenceshown in SE ID Nos. 1 and 2 are generally novel. The highest homology ofthe DNA sequence shown in SEQ ID No. 1 to known protease genes was foundto be 74.7% to the Aspergillus niger acidic proteinase A as determinedfor an overlap of 538 nucleotides. The highest homology to protease IIwas found to be 75.5% to the Aspergillus oryzae aspergillopepsin O asdetermined for an overlap of 343 nucleotides.

In further aspects the invention relates to an expression vectorharbouring a DNA construct of the invention, a cell comprising the DNAconstruct or expression vector and a method of producing an enzymeexhibiting protease activity which method comprises culturing said cellunder conditions permitting the production of the enzyme, and recoveringthe enzyme from the culture.

In a still further aspect the invention relates to an enzyme exhibitingprotease activity, which enzyme is encoded by a DNA construct of theinvention as defined above or produced by the method of the invention.

In a further important aspect the invention relates to an enzyme withprotease activity, which is active at a pH below 7.0 and in the presenceof up to 3% hydrogen peroxide. In the present context, the term “isactive” as used about the enzyme is intended to indicate that the enzymeis capable of hydrolysing a substrate under the above-mentionedconditions, e.g. as described in example 5 herein.

This enzyme of the invention which is active at a pH below 7.0 and inthe presence of up to 3% hydrogen peroxidase and which removes more than80% of the lysozyme from the lenses under the conditions specified inexample 5 is termed the “H₂O₂-stable protease” in the followingdisclosure. This enzyme is believed to be generally novel.

Also, the present invention provides an enzyme with protease activity,which enzyme is active at a pH below 7.0 and which is specific towardsPhe-Val or Lys-Tyr linkages. The term “specific” is intended to indicatethat the enzyme, when the substrate is bovine glucagon, primarilycleaves these linkages.

Protease I and protease II described herein are preferred examples of anenzyme of the invention. The enzymes have been found to be acidproteases, i.e. proteases which has an acid pH optimum.

By the present invention it is possible to provide the protease in ahighly purified form, i.e. greater than 75% pure, and more preferablygreater than 90% pure as determined by SDS gel electrophoresis asdescribed in the Materials and Methods section herein.

In final aspects the invention relates to an enzyme preparationcomprising an enzyme of the invention and the use of the enzyme orenzyme preparation for various purposes in which modification ordegradation of protein-containing substances is desirable.

DETAILED DESCRIPTION OF THE INVENTION

The DNA construct, vector and method of the invention

In the present context the term “analogue” used to define the DNAconstruct of the invention is understood to include any DNA sequencewhich encodes an enzyme with protease activity and which is at least 80%homologous to the DNA sequence shown in SEQ ID No. 1 or 2, respectively.The analogous DNA sequence may be a DNA sequence which hybridizes to thesame probe as the DNA coding for the protease enzyme under the followingconditions: presoaking in 5×SSC and prehybridizing for 1 h at −55° C. ina solution of 5×SSC, 5×Denhardt's solution, 50 mM sodium phosphate, pH6.8, and 50 μg of denatured sonicated calf thymus DNA, followed byhybridization in the same solution supplemented with 50 μCi 32-P-dCTPlabelled probe for 18 h at ˜55° C. followed by washing three times in2×SSC, 0.2% SDS at 55° C. for 30 minutes. The analogous DNA sequence ispreferably at least 90% homologous to the sequence shown in SEQ ID No. 1or 2, preferably at least 95% homologous to said sequence.

The analogous DNA sequence may, e.g., be isolated from another organismor may be one prepared on the basis of the DNA sequence shown in SEQ IDNo. 1 or 2, such as by introduction of nucleotide substitutions which donot give rise to another amino acid sequence of the protease but whichcorrespond to the codon usage of the host organism into which the DNAconstruct is introduced or nucleotide substitutions which do give riseto a different amino acid sequence and therefore, possibly, a differentprotein structure which might give rise to a protease mutant withdifferent properties than the native enzyme. Other examples of possiblemodifications are insertion of one or more nucleotides into thesequence, addition of one or more nucleotides at either end of thesequence, or deletion of one or more nucleotides at either end or withinthe sequence.

Furthermore, it is preferred that the protease encoded by the analogousDNA sequence is immunologically cross-reactive with an antibody raisedagainst a purified protease encoded by the DNA sequence shown in SEQ IDNo. 1 or 2.

The nucleotide probe with which the analogue of the DNA sequence shownin SEQ ID No. 1 can hybridize may, e.g. be prepared on the basis of anyof the following DNA sequences or any combination thereof:

(a) AATTAAGCAT CCTCCATCTT (SEQ ID NO:3) (b) CAAAGCTCAA TCTCGCTAAC (SEQID NO:4) (c) TCCCGCTCTT CTCTCGATCT (SEQ ID NO:5) (d)CATCATCCCA ATAACTCGGA (SEQ ID NO:6) (e) CAAAATGAAG ACCTCTGCTC (SEQ IDNO:7) (f) TCTTGACCGC TGGCCTGTTG (SEQ ID NO:8) (g) GCACCGCTGC TATTGCTGCT(SEQ ID NO:9) (h) CCTCTCACCG CGAAGCGCGC (SEQ ID NO:10) (i)ACGTGCTCGC GCTGCCAAGC (SEQ ID NO:11) (j) TGGCACCAGC CGCAAGAGCA (SEQ IDNO:12) (k) AGGGGGGTCT CAAGCCCGGC (SEQ ID NO:13) (l)ACCCAGCGAG GCCATAACCT (SEQ ID NO:14) (m) GACCGGCTCC AAGAACACCG (SEQ IDNO:15) (n) GAGGTACTCG TCCAACTGGG (SEQ ID NO:16) (o) CCGGCGCCGT GCCAT(SEQ ID NO:17) (p)AATTAAGCAT CCTCCATCTT CAAAGCTCAA TCTCGCTAAC TCCCGCTCTT (SEQ ID NO:18)CTCTCGATCT CATCATCCCA ATAACTCGGA CAAAATGAAG ACCTCTGCTCTCTTGACCGC TGGCCTGTTG GCACCGCTGC TATTGCTGCT CCTCTCACCGCGAAGCGCGC ACGTGCTCGC GCTGCCAAGC TGGCACCAGC CGCAAGAGCAAGGGGGGTCT CAAGCCCGGC ACCCAGCGAG GCCATAACCT GACCGGCTCCAAGAACACCG GAGGTACTCG TCCAACTGGG CCGGCGCCGT GCCAT

These sequences constitute partial sequences of the DNA sequence shownin SEQ ID No. 1 or analogues of such sequences.

The nucleotide probe with which the analogue of the DNA sequence shownin SEQ ID No. 2 can hybridize may, e.g., be prepared on the basis of anyof the following DNA sequences or any combination thereof:

(pl) CTGCTTCTCC TTCTCTTCCT (SEQ ID NO:19) (q) CCTCGTGATA TCTGCTTGAA (SEQID NO:20) (r) CATCTCCTCA TCATGGTCGT (SEQ ID NO:21) (s)CCTCAACAAG GTGCAGCCTT (SEQ ID NO:22) (t) CTTCTGGGTC TGACCACCGC (SEQ IDNO:23) (u) CGCCACTGGT CCCCTGGCCG (SEQ ID NO:24) (v)AGCCGCAGGC TTCTGTCCGG (SEQ ID NO:25) (w) TCAAGAACTT CTCCGTCAAG (SEQ IDNO:26) (x) CAGGTCGAGA AGGCGGGCAG (SEQ ID NO:27) (y)CAAGGGACGT ACCGTTAACC (SEQ ID NO:28) (z) TGCCGGGTCT GTATGCGAAT (SEQ IDNO:29) (aa) GCGCTGGCCA AGTATGGCGC (SEQ ID NO:30) (bb)CCAGGTGCGG CCAGCGTCAA (SEQ ID NO:31) (cc) GGCCGCCGCC GTCAGTGGCA (SEQ IDNO:32) (dd) GCGTCGTGAC CACCCGCAGG CCAACGACG (SEQ ID NO:33) (ee)CTGCTTCTCC TTCTCTTCCT CCTCGTGATA TCTGCTTGAA CATCTCCTCATCATGGTCGT CCTCAACAAG GTGCAGCCTT CTTCTGGGTC TGACCACCGCCGCCACTGGT CCCCTGGCCG AGCCGCAGGC TTCTGTCCGG TCAAGAACTTCTCCGTCAAG CAGGTCGAGA AGGCGGGCAG CAAGGGACGT ACCGTTAACCTGCCGGGTCT GTATGCGAAT GCGCTGGCCA AGTATGGCGC CCAGGTGCGGCCAGCGTCAA GGCCGCCGCC GTCAGTGGCA GCGTCGTGAC CACCCGCAGG CCAACGACG (SEQ IDNO:34)

These sequences constitute partial sequences of the DNA sequence shownin SEQ ID No. 2 or analogues of such sequences.

A DNA sequence of the invention may be isolated by a general methodinvolving

cloning, in suitable vectors, a DNA library from Aspergillus aculeatus,

transforming suitable yeast host cells with said vectors,

culturing the host cells under suitable conditions to express any enzymeof interest encoded by a clone in the DNA library, and

screening for positive clones by determining any protease activity ofthe enzyme produced by such clones.

A more detailed description of this screening method is given in Example1 below and in WO 93/11249, the contents of which is hereby incorporatedby reference.

The DNA sequence coding for the enzyme may for instance be isolated byscreening a cDNA library of Aspergillus aculeatus, e.g. strain CBS101.43, publicly available from the Centraalbureau voorSchimmelcultures, Delft, NL, and selecting for clones expressing theappropriate enzyme activity (i.e. protease activity as defined by theability of the enzyme to hydrolyse peptide bonds in proteins andpeptides). The appropriate DNA sequence may then be isolated from theclone by standard procedures, e.g. as described in Example 1.

It is expected that a DNA sequence coding for a homologous enzyme, i.e.an analogous DNA sequence, may be derived by similarly screening a cDNAlibrary of another microorganism, in particular a fungus, such as astrain of another Aspergillus sp., in particular a strain of A.aculeatus or A. niger, a strain of a Trichoderma sp., in particular astrain of T. harzianum, or T. reesie, a strain of a Fusarium sp., inparticular a strain of F. oxysporum, a strain of Rhizopus sp., e.g. R.niveus, a strain of Schytalidum sp., or a strain of a Humicola sp.

Alternatively, the DNA sequence of the invention may, in accordance withwell-known procedures, conveniently be isolated from DNA from anappropriate organism by use of synthetic oligonucleotide probes preparedon the basis of a DNA sequence disclosed herein. For instance, asuitable oligonucleotide probes may be prepared on the basis of any ofthe partial nucleotide sequences shown above.

The DNA sequence may subsequently be inserted into a recombinantexpression vector. This may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e. a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the protease should be operablyconnected to a suitable promoter and terminator sequence. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. The procedures used toligate the DNA sequences coding for the protease, the promoter and theterminator, respectively, and to insert them into suitable vectors arewell known to persons skilled in the art (cf., for instance, Sambrook etal., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.,1989).

The host cell which is transformed with the DNA sequence encoding theenzyme of the invention is preferably a eukaryotic cell, in particular afungal cell such as a yeast or filamentous fungal cell. In particular,the cell may belong to a species of Aspergillus, most preferablyAspergillus oryzae or Aspergillus niger. Fungal cells may be transformedby a process involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall in a manner knownper se. The use of Aspergillus oryzae as a host microorganism isdescribed in EP 238 023 (of Novo Noridisk A/S), the contents of whichare hereby incorporated by reference. The host cell may also be a yeastcell, e.g. a strain of Saccharomyces, in particular Saccharomycescerevisiae, Saccharomyces kluyveri or Saccharomyces uvarum, a strain ofSchizosaccaromyces sp., such as Schizosaccharomyces pombe, a strain ofHansenula sp., Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, orKluyveromyces sp. such as Kluyveromyces lactis.

In a still further aspect, the present invention relates to a method ofproducing an enzyme with protease activity, wherein a suitable host celltransformed with a DNA construct of the invention encoding the enzyme iscultured under conditions permitting the production of the enzyme, andthe resulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed protease may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

The enzyme of the invention

Preferably, the protease of the invention (e.g. The H₂O₂-stable or theLys-Tyr or Phe-Val specific protease) is active at a pH in the range of2-7, such as at a pH below 6.0, e.g. in the range of 2-6, and mostpreferably in the pH range of 4-6.

It will be understood that both acidic proteases are active in thepresence of 0.01-5% hydrogen peroxide, such as 0.1-5%, 05.-4%, 1-4% of2-3% hydrogen peroxide and are applicable to contact lens cleaning.

A preferred example of an H₂O₂-stable protease of the invention is theenzyme encoded by the DNA sequence shown in SEQ ID No. 1 or an analoguethereof as defined above, which is at least 80% homologous to said DNAsequence.

A preferred example of the Lys-Tyr or Phe-Val specific protease of theinvention is the enzyme encoded by the DNA sequence shown in SEQ ID No.2 or an analogue thereof as defined above, which is at least 80%homologous to said DNA sequence.

The enzyme of the invention is preferably immunologically reactive withan antibody raised against a purified protease derived from Aspergillusaculeatus, CBS 101.43 and being encoding by the DNA sequence shown inSEQ ID No. 1 or 2. In the present context, the term “derived from” isintended not only to indicate a protease produced by strain CBS 101.43,but also a protease encoded by a DNA sequence isolated from strain CBS101.43 and produced in a host organism transformed with said DNAsequence.

While the H₂O₂-stable protease and the Lys-Tyr or Phe-Val specificprotease of the invention both were obtained from a strain of the fungalspecies Aspergillus aculeatus, it is contemplated that such enzymes areobtainable from other organisms as well, in particularly microorganisms.

Thus, the enzyme of the invention is preferably obtainable from abacterium or a fungus such as a strain of Aspergillus, Rhizopus,Trichoderma, e.g. T. reesei or T. harzianum, Penicillium, Fusarium,Schytalidium or Humicola, e.g. H. insolens or H. lanuginosa, or a strainof Bacillus.

Examples of Aspergillus sp. include A. niger, A. oryzae or A. aculeatus,such as A. aculeatus CBS 101.43.

In a still further aspect, the present invention relates to an enzymepreparation useful for the degradation or modification of proteasecontaining materials, said preparation being enriched in an enzymeexhibiting protease activity as described above.

The enzyme preparation having been enriched with an enzyme of theinvention may e.g. be an enzyme preparation comprising multipleenzymatic activities, in particular an enzyme preparation comprisingmultiple plant cell wall degrading enzymes such as Pectinex®, PectinexUltra SP®, Gamanase, Celluclast or Celluzyme or protease and/orexopeptidase-containing enzyme preparations such as Neutrase®, Alcalase®or Flavourzyme® (all available from Novo Nordisk A/S). In the presentcontext, the term “enriched” is intended to indicate that the proteaseactivity of the enzyme preparation has been increased, e.g. with anenrichment factor of at least 1.1, conveniently due to addition of anenzyme of the invention prepared by the method described above.

Alternatively, the enzyme preparation enriched in an enzyme exhibitingprotease activity may be one which comprises an enzyme of the inventionas the major enzymatic component, e.g. a mono-component enzymepreparation.

The enzyme preparation may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry preparation. Forinstance, the enzyme preparation may be in the form of a granulate or amicrogranulate. The enzyme to be included in the preparation may bestabilized in accordance with methods known in the art.

The enzyme preparation according to the invention may be used as anagent for degradation or modification of plant cell walls. Someproteins, like extensins, are components of plant cell walls. Proteaseswill therefore facilitate the degradation or modification of plant cellwalls. Such a protease containing plant cell wall degrading enzymepreparation can be used for many different applications like extractionof oil from plant sources like olives and rape or for production ofjuice from different fruits like apples, pears and citrus.

The enzyme preparation may additionally contain one or more other plantcell wall degrading enzymes such as a pectin lyase, pectate lyase,endoglucanase, arabinanase, xylanase, glucanase, galactanase, mannanase,α-galactosidase, rhamnogalacturonase, pectin acetylesterase,polygalacturonase, protease, exo-peptidase or pectin methylesterase. Thepreparation may further contain one or more enzymes exhibitingexo-activity on the same substrates as the above-mentioned endo-enzymes.The proteases according to the invention work at the same pH andtemperature conditions as many other cell wall degrading enzymes, andare thereby particular well suited for such applications.

The additional enzyme(s) may be producible by means of a microorganismbelonging to the genus Aspergillus, preferably Aspergillus niger,Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae, orTrichoderma.

The protease enzyme preparation according to the invention may also beused in the wine industry, to prevent haze or to dissolve haze, asproteins often take part in the undesirable haze formation. Theproteases according to the invention are active under the conditionspresent under fermentation and maturation of wine, and they aretherefore particular useful for this application.

The enzyme or enzyme preparation of the invention may be used in baking,e.g. in order to weaken the gluten components of flour so as to obtain asoftening of so-called hard four. The use of weak flour is important forthe preparation of dough which must be very extensible and not elastic,e.g. in the preparation of extruded baked products, biscuits and otherproducts which must keep their original shape during transport andbaking. The proteases of the invention constitute a desirablealternative to conventionally used agents for weakening of flour, suchas sodium metabisulphite (SMS). The use of SMS is considered to beundesirable because of potential health risks.

The protease preparation may also be used in the food and feed industryto improve the digestibility of proteins. For instance, the enzyme orenzyme preparation may be added to animal feed or may be used to processanimal feed, in particular feed for piglets or poultry. Thereby, thedigestibility of components of the feed may be increased resulting in animproved growth rate and efficiency of feed utilisation of the animals,cf. Brenes et al. (1993).

Further the enzyme or enzyme preparation of the invention may be usefulto make protein hydrolysates from, e.g., vegetable proteins like soy,pea, lupin or rape seed protein, milk like casein, meat proteins, orfish proteins. The protease may be used for protein hydrolysates toimprove the solubility, consistency, taste, or fermentability, to reduceantigenicity or for other purposes to make food, feed or dedicalproducts. The protease may be used alone or together with otherproteases or together with other enzymes like exopeptidases. The use ofthe protease of the invention together with exopeptidase rich enzymepreparations will improve the taste of the protein hydrolysates.

The protease preparation may also be used to modify proteins, likereducing viscosity caused or partially caused by proteins. Suchviscosity problems are known in the processing of different proteincontaining plant materials like soya and peas.

Furthermore, the enzyme or enzyme preparation may be used in theprocessing of fish or meat, e.g. to change texture and/or viscosity.

The protease preparation may also be used to facilitate fermentativeprocesses, like yeast fermentation of barley, malt and other rawmaterials for the production of e.g. beer.

Furthermore, the enzyme or enzyme preparation of the invention may beuseful in the leather industry e.g. to remove hairs from hides. The lowpH optimum of the protease is an advantage as the subsequent tanning ofthe hides is carried under acid conditions. The protease preparation isuseful for production of peptides from proteins, where it is advantagesto use a cloned enzyme essentially free from other proteolyticactivities.

Further the protease preparation can be used to degrade protein in orderto facilitate purification of or to upgrade different products, like inpurification or upgrading of gums, like guar gum, xanthan gum, degummingof silk, or improvement of the quality of wool.

Due to the stability towards hydrogen peroxide both proteases of theinvention are of particular use for cleaning of contact lenses and otherapplications involving the use of hydrogen peroxide, in whichprotein-containing material is to be removed.

For the above uses, the dosage of the enzyme preparation of theinvention and other conditions under which the preparation is used maybe determined on the basis of methods known in the art.

The invention is further described in the accompanying drawing in which

FIG. 1 the pH activity profiles of Protease I and II, respectively,

FIG. 2 the temperature-activity profiles of Protease I and II,respectively,

FIGS. 3 and 4 the pH stability of Protease I and II, respectively,

FIGS. 5 and 6 the temperature stability of Protease I and II,respectively,

FIG. 7 the performance of various proteases in the cleaning of contactlenses, and

FIG. 8 the gluten stretching effect of an enzyme of the invention.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

MATERIALS AND METHODS

Donor Organism

mRNA was isolated from Aspergillus aculeatus, CBS 101.43, grown in asoy-containing fermentation medium with agitation to ensure sufficientaeration. Mycelia were harvested after 3-5 days' growth, immediatelyfrozen in liquid nitrogen and stored at −80° C.

Yeast Strains

The Saccharomyces cerevisiae strain used was yNG231 (MAT alpha, leu2,ura3-52, his 4-539, pep4-delta 1, cir+) or JG169 (MATα; ura 3-52; leu2-3, 112; his 3-D200; pep 4-113; prc1::HIS3; prb1:: LEU2; cir+).

Plasmids

The expression plasmid pYHD17 containing the yeast TPI promoter wasprepared from the commercially available plasmid pYES II (Invitrogen).The plasmid and the construction thereof is further described in WO93/11249, the contents of which is hereby incorporated by reference.

The Aspergillus expression vector pHD414 is a derivative of the plasmidp775 (described in EP 238 023). The construction of pHD414 is furtherdescribed in WO 93/11249. pHD414 contains the A. niger glucoamylaseterminator and the A. oryzae TAKA amylase promoter.

Extraction of Total RNA

The total RNA was prepared by extraction with guanidinium thiocyanatefollowed by ultracentrifugation through a 5.7 M CsCl cushion essentiallyas described by Chirgwin et al., 1979 and in WO 93/11249.

Isolation of poly(A)⁺RNA

The poly(A)⁺RNAs were isolated by oligo(dT)-cellulose affinitychromatography (Aviv & Leder, 1972). Typically, 0.2 g of oligo(dT)cellulose (Boehringer Mannheim) was preswollen in 10 ml of 1×columnloading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS),loaded onto a DEPC-treated, plugged plastic column (Poly PrepChromatography Column, Bio Rad), and equilibrated with 20 ml 1×loadingbuffer. The total RNA was heated at 65° C. for 8 min., quenched on icefor 5 min, and after addition of 1 vol 2×column loading buffer to theRNA sample loaded onto the column. The eluate was collected and reloaded2-3 times by heating the sample as above and quenching on ice prior toeach loading. The oligo(dT) column was washed with 10 vols of 1×loadingbuffer, then with 3 vols of medium salt buffer (20 mM Tris-Cl, pH 7.6,0.1 M NaCl, 1 mM EDTA, 0.1% SDS), followed by elution of the poly(A)⁺RNA with 3 vols of elution buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA,0.05% SDS) preheated to +65° C., by collecting 500 μl fractions. TheOD₂₆₀ was read for each collected fraction, and the mRNA containingfractions were pooled and ethanol precipitated at −20° C. for 12 h. Thepoly(A)⁺ RNA was collected by centrifugation, resuspended in DEPC-DIWand stored in 5-10 μg aliquots at −80° C.

Northern Blot Analysis

The poly(A)⁺ RNAs (5 μg/sample) from various mycelia wereelectrophoresed in 1.2 agarose-2.2 M formaldehyde gels (Sambrook et al.,1989) and blotted to nylon membranes (Hybond-N, Amersham) with 10×SSC(Sambrook et al., 1989) as transfer buffer. Three random-primed(Feinberg & Vogelstein, 1983) ³²P-labeled cDNA probes were used inindividual hybridizations: 1) a 1.3 kb Not I-Spe I fragment forpolygalacturonase I from A. aculeatus, 2) a 1.3 kb Not I-Spe I fragmentencoding endoglucanase I from A. aculeatus and 3) a 1.2 kb Eag Ifragment coding for galactanase I from A. aculeatus. Northernhybridizations were carried out in 5×SSC (Sambrook et al., 1989),5×Denhardt's solution (Sambrook et al., 1989), 0.5% SDS (w/v) and 100μg/ml denatured salmon sperm DNA with a probe concentration of ca. 2ng/ml for 16 h at 65° C. followed by washes in 5×SSC at 65° C. (2×15min), 2×SSC, 0.5% SDS (1×30 min), 0.2×SSC, 0.5% SDS (1×30 min), and5×SSC (2×15 min). After autoradiography at −80° C. for 12 h, the probe#1 was removed from the filter according to the manufacturer'sinstructions and rehybridized with probe #2, and eventually with probe#3. The RNA ladder from Bethesda Research Laboratories was used as asize marker.

cDNA Synthesis

First Strand Synthesis

Double-stranded cDNA was synthesized from 5 μg of A. aculeatus poly(A)⁺RNA by the RNase H method (Gubler & Hoffman 1983, Sambrook et al., 1989)using the hair-pin modification. The poly(A)⁺RNA (5 μg in 5 μl ofDEPC-treated water) was heated at 70° C. for 8 min., quenched on ice,and combined in a final volume of 50 μl with reverse transcriptasebuffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT,Bethesda Research Laboratories) containing 1 mM each dNTP (Pharmacia),40 units of human placental ribonuclease inhibitor (RNasin, Promega), 10μg of oligo(dT)₁₂₋₁₈ primer (Pharmacia) and 1000 units of SuperScript IIRNase H- reverse transcriptase (Bethesda Research Laboratories).First-strand cDNA was synthesized by incubating the reaction mixture at45° C. for 1 h.

Second Strand Synthesis

After synthesis 30 μl of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA was added, andthe mRNA:cDNA hybrids were ethanol precipitated for 12 h at −20° C. byaddition of 40 μg glycogen carrier (Boehringer Mannheim) 0.2 vols 10 MNH₄Ac and 2.5 vols 96% EtOH. The hybrids were recovered bycentrifugation, washed in 70% EtOH, air dried and resuspended in 250 μlof second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2,10 mM (NH₄)₂SO₄, 16 μM βNAD⁺) containing 100 μM each dNTP, 44 units ofE. coli DNA polymerase I (Amersham), 6.25 units of RNase H (BethesdaResearch Laboratories) and 10.5 units of E. coli DNA ligase (New EnglandBiolabs). Second strand cDNA synthesis was performed by incubating thereaction tube at 16° C. for 3 h, and the reaction was stopped byaddition of EDTA to 20 mM final concentration followed by phenolextraction.

Mung Bean Nuclease Treatment

The double-stranded (ds) cDNA was ethanol precipitated at −20° C. for 12h by addition of 2 vols of 96% EtOH, 0.1 vol 3 M NaAc, pH 5.2, recoveredby centrifugation, washed in 70% EtOH, dried (SpeedVac), and resuspendedin 30 μl of Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl,1 mM ZnSO4, 0.35 mM DTT, 2% glycerol) containing 36 units of Mung beannuclease (Bethesda Research Laboratories). The single-stranded hair-pinDNA was clipped by incubating the reaction at 30° C. for 30 min,followed by addition of 70 μl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenolextraction, and ethanol precipitation with 2 vols of 96% EtOH and 0.1vol 3M NaAc, pH 5.2 at −20° C. for 12 h.

Blunt-ending with T4 DNA Polymerase

The ds cDNA was blunt-ended with T4 DNA polymerase in 50 μl of T4 DNApolymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1mM DTT) containing 0.5 mM each dNTP and 7.5 units of T4 DNA polymerase(Invitrogen) by incubating the reaction mixture at +37° C. for 15 min.The reaction was stopped by addition of EDTA to 20 mM finalconcentration, followed by phenol extraction and ethanol precipitation.

Adaptor Ligation and Size Selection

After the fill-in reaction the cDNA was ligated to non-palindromic BstXI adaptors (1 μg/μl, Invitrogen) in 30 μl of ligation buffer (50 mMTris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 μg/ml bovine serumalbumin) containing 600 pmol BstX I adaptors and 5 units of T4 ligase(Invitrogen) by incubating the reaction mix at +16° C. for 12 h. Thereaction was stopped by heating at +70° C. for 5 min, and the adaptedcDNA was size-fractionated by agarose gel electrophoresis (0.8%HSB-agarose, FMC) to separate unligated adaptors and small cDNAs. ThecDNA was size-selected with a cut-off at 0.7 kb, and the cDNA waselectroeluted from the agarose gel in 10 mM Tris-Cl, pH 7.5, 1 mM EDTAfor 1 h at 100 volts, phenol extracted and ethanol precipitated at −20°C. for 12 h as above.

Construction of cDNA Libraries

The adapted, ds cDNA was recovered by centrifugation, washed in 70% EtOHand resuspended in 25 ml DIW. Prior to large-scale library ligation,four test ligations were carried out in 10 μl of ligation buffer (sameas above) each containing 1 μl ds cDNA (reaction tubes #1-#3), 2 unitsof T4 ligase (Invitrogen) and 50 ng (tube #1), 100 ng (tube #2) and 200ng (tubes #3 and #4) Bst XI cleaved yeast expression vector either pYES2.0 vector Invitrogen or yHD17). The ligation reactions were performedby incubation at +16° C. for 12 h, heated at 70° C. for 5 min, and 1 μlof each ligation electroporated (200 Ω, 2.5 kV, 25 μF) to 40 μlcompetent E. coli 1061 cells (OD600=0.9 in 1 liter LB-broth, washedtwice in cold DIW, once in 20 ml of 10% glycerol, resuspended in 2 ml10% glycerol). After addition of 1 ml SOC to each transformation mix,the cells were grown at +37° C. for 1 h, 50 μl plated on LB+ampicillinplates (100 μg/ml) and grown at +37° C. for 12 h.

Using the optimal conditions a large-scale ligation was set up in 40 μlof ligation buffer containing 9 units of T4 ligase, and the reaction wasincubated at +16° C. for 12 h. The ligation reaction was stopped byheating at 70° C. for 5 min, ethanol precipitated at −20° C. for 12 h,recovered by centrifugation and resuspended in 10 μl DIW. One μlaliquots were transformed into electrocompetent E. coli 1061 cells usingthe same electroporation conditions as above, and the transformed cellswere titered and the library plated on LB+ampicillin plates with5000-7000 c.f.u./plate. To each plate was added 3 ml of medium. Thebacteria were scraped off, 1 ml glycerol was added and stored at −80° C.as pools. The remaining 2 ml were used for DNA isolation. If the amountof DNA was insufficient to give the required number of yeasttransformants, large scale DNA was prepared from 500 ml medium (TB)inoculated with 50 μl of −80° C. bacterial stock propagated overnight.

Construction of Yeast Libraries

To ensure that all the bacterial clones were tested in yeast, a numberof yeast transformants 5 times larger than the number of bacterialclones in the original pools was set as the limit.

One μl aliquots of purified plasmid DNA (100 ng/μl) from individualpools were electroporated (200 Ω, 1.5 kV, 25 μF) into 40 μl competent S.cerevisiae JG 169 cells (OD600=1.5 in 500 ml YPD, washed twice in coldDIW, once in cold 1 M sorbitol, resuspended in 0.5 ml 1 M sorbitol,Becker & Guarante, 1991). After addition of 1 ml 1M cold sorbitol, 80 μlaliquots were plated on SC+glucose−uracil to give 250-400 c.f.u./plateand incubated at 30° C. for 3-5 days.

Isolation of a cDNA Gene for Expression in Aspergillus

One or more of protease-producing colonies were inoculated into 20 mlYNB-1 broth in a 50 ml glass test tube. The tube was shaken for 2 daysat 30° C. The cells were harvested by centrifugation for 10 min. at 3000rpm.

The cells were resuspended in 1 ml 0.9 M sorbitol, 0.1 M EDTA, pH 7.5.The pellet was transferred to an Eppendorf tube, and spun for 30 secondsat full speed. The cells were resuspended in 0.4 ml 0.9 M sorbitol, 0.1M EDTA, 14 mM β-mercaptoethanol. 100 μl 2 mg/ml Zymolase was added, andthe suspension was incubated at 37° C. for 30 minutes and spun for 30seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE. 90 μlof (1.5 ml 0.5 M EDTA pH 8.0, 0.6 ml 2 M Tris-Cl pH 8.0, 0.6 ml 10% SDS)was added, and the suspension was incubated at 65° C. for 30 minutes. 80μl 5 M KOAc was added, and the suspension was incubated on ice for atleast 60 minutes and spun for 15 minutes at full speed. The supernatantwas transferred to a fresh tube which was filled with EtOH (room temp.)followed by thorough but gentle mixing and spinning for 30 seconds. Thepellet was washed with cold 70% ETOH, spun for 30 seconds and dried atroom temperature. The pellet was resuspended in 50 μl TE and spun for 15minutes. The supernatant was transferred to a fresh tube. 2.5 μl 10mg/ml RNase was added, followed by incubation at 37° C. for 30 minutesand addition of 500 μl isopropanol with gentle mixing. The mixture wasspun for 30 seconds, and the supernatant was removed. The pellet wasrinsed with cold 95% EtOH and dried at room temperature. The DNA wasdissolved in 50 μl water to a final concentration of approximately 100μl/ml.

Transformation of Aspergillus oryzae or Aspergillus niger (GeneralProcedure)

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) is inoculated with spores of A. oryzae or A.niger and incubated with shaking at 37° C. for about 2 days. Themycelium is harvested by filtration through miracloth and washed with200 ml of 0.6 M MgSO₄. The mycelium is suspended in 15 ml of 1.2 MMgSO₄. 10 mM NaH₂PO₄, pH=5.8. The suspension is cooled on ice and 1 mlof buffer containing 120 mg of Novozym® 234, batch 1687 is added. After5 minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added and incubationwith gentle agitation continued for 1.5-2.5 hours at 37° C. until alarge number of protoplasts is visible in a sample inspected under themicroscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayered with 5 ml of 0.6 M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation is performed for 15 minutes at 100 gand the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5. 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally the protoplasts areresuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC. Protoplasts are mixed with p3SR2 (an A. nidulansamdS gene carrying plasmid). The mixture is left at room temperature for25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM CaCl₂ and 10 mMTris-HCl, pH=7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution is added and carefully mixed. The mixture isleft at room temperature for 25 minutes, spun at 2500 g for 15 minutesand the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one moresedimentation the protoplasts are spread on the appropriate plates.Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113(1966) 51-56) containing 1.0 M sucrose, pH=7.0, 10 mM acetamide asnitrogen source and 20 mM CsCl to inhibit background growth. Afterincubation for 4-7 days at 37° C. spores are picked and spread forsingle colonies. This procedure is repeated and spores of a singlecolony after the second reisolation is stored as a defined transformant.

Immunological Cross-reactivity

Antibodies to be used in determining immunological cross-reactivity maybe prepared by use of a purified protease. More specifically, antiserumagainst a protease of the invention may be raised by immunizing rabbits(or other rodents) according to the procedure described by N. Axelsen etal. in: A Manual of Quantitative Immunoelectrophoresis, BlackwellScientific Publications, 1973, Chapter 23, or A. Johnstone and R.Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pp. 27-31). Purified immunoglobulins may beobtained from the antisera, for example by salt precipitation ((NH₄)₂SO₄), followed by dialysis and ion exchange chromatography, e.g. onDEAE-Sephadex. Immunochemical characterization of proteins may be doneeither by Outcherlony double-diffusion analysis (O. Ouchterlony in:Handbook of Experimental Immunology (D. M. Weir, Ed.), BlackwellScientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), orby rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

Media

YPD: 10 g yeast extract, 20 g peptone, H₂O to 810 ml. Autoclaved, 90 ml20% glucose (sterile filtered) added.

10×Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 gNaOH, H₂O ad 1000 ml, sterile filtered.

SC-URA: 90 ml 10×Basal salt, 22.5 ml 20% casamino acids, 9 ml 1%tryptophan, H₂O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20%glucose or 20% galactose added.

SC—H broth: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan. Autoclaved for 20 min. at 121° C. After autoclaving,10 ml of a 30% galactose solution, 5 ml of a 30% glucose solution and0.4 ml of a 5% threonine solution were added per 100 ml medium.

SC—H agar: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/lsuccinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins,0.1 g/l tryptophan, and 20 g/l agar (Bacto). Autoclaved for 20 min. at121° C. After autoclaving, 55 ml of a 22% galactose solution and 1.8 mlof a 5% threonine solution were added per 450 ml agar.

YNB-1 agar: 3.3 g/l KH₂PO₄, 16.7 g/l agar, pH adjusted to 7. Autoclavedfor 20 min. at 121° C. After autoclaving, 25 ml of a 13.6% yeastnitrogen base without amino acids, 25 ml of 40% glucose solution, 1.5 mlof a 1% L-leucine solution and 1.5 ml of a 1% histidine solution wereadded per 450 ml agar.

YNB-1broth: Composition as YNB-1 agar, but without the agar.

FG-4-Agar: 35 g/L agar, 30 g/L Sor bean meal, 15 g/L maltodextrin(Glucidex 6), 5 g/L Bacto pepton, pH 7. Autoclaved 40 min at 121° C.

FG-4 medium: 30 g/L Soy bean meal, 15 g/L maltodextrin (Glucidex 6), 5g/L Bacto peptone. Autoclaved 40 min at 121° C.

MDU-2 medium: 45 g/L maltose, 1 g/L MgSO₄-7 H₂O, 1 g/L NaCl, 2 g/LK₂SO₄, 12 g/L KH₂PO₄, 0.1 ml/L Pluronic 61 L, 0.5 ml/L Trace metalsolution. pH 5.0. Autoclaved 20 min at 121° C. 15 ml/L 50% sterilefiltered urea is added after autoclaving.

Casein overlayer gel: 1% agarose, 0.5% casein in a buffer with a pH of5.5. The gel was boiled and then cooled to 55° C. before the overlayerwas poured onto agar plates.

Fed Batch Fermentation

The medium used for fed-batch fermentation of protease I or II by A.oryzae comprised maltodextrin as a carbon source, urea as a nitrogensource and yeast extract.

The fed batch fermentation was performed by innoculating a shake flaskculture of the A. oryzae host cells in question into a medium comprising3.5% of the carbon source and 0.5% of the nitrogen source. After 24hours of cultivation at pH 5.0 and 34° C. the continuous supply ofadditional carbon and nitrogen sources were initiated. The carbon sourcewas kept as the limiting factor and it was secured that oxygen waspresent in excess. The fed batch cultivation was continued for 4 days,after which the enzymes could be recovered.

Characterization of Enzymes

Proteolytic Activity

1 hemoglobin protease unit (hpu) is defined as the amount of enzymeliberating 1 millimole of primary amino groups (determined by comparisonwith a serine standard) per minute under standard conditions asdescribed below:

A 2% (w/v) solution of hemoglobin (bovine, supplied by Sigma) isprepared with the Universal Buffer described by Britton and Robinson, J.Chem. Soc., 1931, p. 1451), adjusted to a pH of 5.5. 2 ml of thesubstrate solution are pre-incubated in a water bath for 10 min at 25°C. 1 ml of an enzyme solution containing b g/ml of the enzymepreparation, corresponding to about 0.2-0.3 hpu/ml of the UniversalBuffer (pH 5.5) is added. After 30 min. of incubation at 25° C., thereaction is terminated by the addition of a quenching agent (5 ml of asolution containing 17.9 g of trichloroacetic acid, 29.9 g of sodiumacetate and 19.8 g of acetic acid made up to 500 ml with deionizedwater). A blank is prepared in the same way as the test solution withthe exception that the quenching agent is added prior to the enzymesolution. The reaction mixtures are kept for 20 min. in a water bathafter which they are filtered through Whatman 42 paper filters.

Primary amino groups are determined by their colour development witho-phthaldialdehyde (OPA), as follows: 7.62 g of disodium tetraboratedecahydrate and 2.0 g of sodium dodecylsulfate are dissolved in 150 mlof water. 160 mg of OPA dissolved in 4 ml of methanol were then addedtogether with 400 μl of β-mercaptoethanol after which the solution ismade up to 200 ml with water. To a 3 ml of the OPA reagent are added 400μl of the filtrates obtained above, with mixing. The optical density(OD) at 340 nm is measured after about 5 min. The OPA test is alsoperformed with a serine standard containing 10 mg of serine in 100 ml ofUniversal Buffer (pH 5.5). The buffer alone is used as a blank. Theprotease activity is calculated from the OD measurements by means of thefollowing formula:$\quad {{{{hpu}/{ml}}\quad {enzyme}\quad {{solution}:\quad {\frac{\left( {{OD}_{l} - {OD}_{b}} \right) \times C_{ser} \times Q}{\left( {{OD}_{ser} - {OD}_{B}} \right) \times {MW}_{ser} \times t_{i}}\quad {{hpu}/g}{\quad \quad}{of}\quad {enzyme}{\quad \quad}{preparation}}}} = {{hpu}/{{ml}:\quad b}}}\quad$

wherein OD_(t), OD_(b), OD_(ser) and OD_(β) is the optical density ofthe test solution, blank, serine standard and buffer, respectively,c_(ser) is the concentration of serine (mg/ml) in the standard (in thiscase 0.1 mg/ml), and MW_(ser) is the molecular weight of serine(105.09). Q is the dilution factor for the enzyme solution (in this case8) and t_(i) is the incubation time in minutes (in this case 30minutes).

Inhibition

The following inhibitors were tested:

Pepstatin (aspartic acid inhibitor) (1 mM)

PMSF (serine protease inhibitor) (0.1%)

PEFABLOC (serine protease inhibitor) (0.1%)

EDTA (metallo protease inhibitor) (0.1M)

all available from Sigma except for PEFABLOC which is available fromPentapharm, Basel, Switzerland.

Residual activity was determined as HPU/l at pH 5.5.

pH activity profiles were determined as HPU/l at different pH values(4-8).

pH-stability was determined by letting an enzyme solution (0.3 HPU/l)stand for 30, 60 and 120 minutes, respectively, at 50° C. and differentpH-values (4-5-6-7-8) and measure proteolytic activity before and afterstanding.

Temperature activity profiles were determined as HPU/l at differenttemperatures (15-70° C.).

Temperature stability was determined by letting an enzyme solution (0.3HPU/l) stand for 30, 60 and 120 minutes at pH 5 and at differenttemperatures (25-40-50-60° C.), and measure proteolytic activity beforeand after standing.

SDS gel electrophoresis and isoelectric focusing was carried out on thePhast-System from Pharmacia using a Gradient 8-25 and the IEF 3-9,respectively, according to the manufacturers instructions.

Specificity

The specificity of proteases of the invention is determined as follows:

0.5 ml of 1 mg/ml human insulin or bovine glucagon in Universal Buffer,pH 5.5 (vide supra), and 75 μl of protease I and II, respectively, (0.6hpu/l) in the same buffer were incubated for 120 min. at 37° C. Thereaction was terminated by adding 50 μl 1N hydrochloric acid.

The insulin or glucagon molecule was cleaved into a number of peptidefragments. These were separated and isolated by reverse phase HPCL usinga suitable C-18 column (Hibar LiChrosorb RP-18, 5 μm particles providedby Merck AG, Darmstadt, FRG). The fragments were eluted with thefollowing solvents:

A. 0.2 M sodium sulfate and 0.1 M phosphoric acid, pH 2.5;

B. Acetonitrile/water, 50%;

on a linear gradient of from 90% A/10% B to 80% A/20% B for 0-5 min. andsubsequently for 50 min. with 80% A/20% B. The isolated fragments weresubjected to amino acid sequencing by automated Edman degradation, usingan Applied Biosystems (Foster City, Calif., USA) Model 470A gas-phasesequencer, and the phenylthiohydantoin (PTH-) amino acids were analyzedby high performance liquid chromatography as described by L. Thim etal., “Secretion of human insulin by a transformed yeast cell”, FEBSLetters 212(2), 1987, p.307, whereby the cleavage sites in the insulinor glucagon molecule were identified.

EXAMPLE 1

A library from A. aculeatus consisting of approx. 1.5×10⁶ individualclones in 150 pools was constructed.

DNA was isolated from 20 individual clones from the library andsubjected to analysis for cDNA insertion. The insertion frequency wasfound to be >90% and the average insert size was approximately 1400 bp.

DNA from some of the pools was transformed into yeast, and 50-100 platescontaining 200-500 yeast colonies were obtained from each pool. After3-5 days of growth, the agar plates were replica plated onto severalsets of agar plates. One set of plates was then incubated for 2-4 daysat 30° C. and overlayered with a casein overlayer gel for detection ofprotease activity. After incubation overnight at 30° C.,protease-positive colonies were identified as colonies surrounded by awhite halo.

Cells from enzyme-positive colonies were spread for single colonyisolation on agar, and an enzyme-producing single colony was selectedfor each of the protease-producing colonies identified.

The positive clones were obtained as single colonies, the cDNA insertswere amplified directly from the yeast colony using biotinylatedpolylinker primers, purified by magnetic beads (Dynabead M-280, Dynal)system and characterized individually by sequencing the 5′-end of eachcDNA clone using the chain-termination method (Sanger et al., 1977) andthe Sequenase system (United States Biochemical). The DNA sequences oftwo enzyme genes are shown in SEQ ID Nos. 1 and 2, respectively.

Subsequently, the cDNA encoding the protease was isolated for expressionin Aspergillus as described above and transformed into E. coli usingstandard procedures. Two E. coli colonies were isolated from each of thetransformations and analysed with the restriction enzymes HindIII andXbaI which excised the DNA insert. DNA from one of these clones wasretransformed into yeast strain JG169.

The DNA sequences of several of the positive clones were determined. TwoDNA sequences encoding a protease are shown in SEQ ID Nos. 1 and 2,respectively.

EXAMPLE 2

In order to express the genes in Aspergillus, cDNA is isolated from oneor more representatives of each family using the above describedprocedure by digestion with HindIII/XbaI or other appropriaterestriction enzymes, size fractionation on a gel and purification andsubsequently ligated to pHD414, resulting in the plasmids pA1P1 andpA1P2. After amplification in E. coli, the plasmids are transformed intoA. oryzae or A. niger according to the general procedure describedabove.

Test of A. oryzae Transformants

Each of the transformants was inoculated in the center of a Petri dishwith FG-4 agar. After 5 days of incubation at 30° C. 4 mm diameter plugswere removed from the center of the colonies by means of a corkscrew.The plugs were embedded in a casein overlayer gel, containing 0.5%casein and 1% agarose in a buffer with a pH of 5.5, and incubatedovernight at 40° C. The protease activity was identified as describedabove. Some of the transformants had halos which were significantlylarger than the Aspergillus oryzae background. This demonstratesefficient expression of protease in Aspergillus oryzae. The 8transformants with the highest protease activity were selected andinoculated and maintained on YPG-agar.

Each of the 8 selected transformants were inoculated from YPG-agarslants on 500 ml shake flask with FG-4 and MDU-2 media. After 3-5 daysof fermentation with sufficient agitation to ensure good aeration, theculture broths were centrifuged for 10 minutes at 2000 g and thesupernatants were analyzed.

A volume of 15 μl of each supernatant was applied to 4 mm diameter holespunched out in a casein overlayer gel (25 ml in a 13 cm diameter Petridish). The protease activity was identified by the formation of a whitehalo on incubation.

Fed batch fermentation

Subsequently, protease I and II, respectively, were produced by fedbatch fermentation of A. oryzae expressing the enzyme using theprocedure described above.

EXAMPLE 3 Characterization of Protease I and II

The supernatant resulting from the fed batch fermentation above was usedfor the characterization performed as described in the Materials andMethods section above. Proteolytic activity was measured as HPU/l at pH5.5 using the above described procedure.

Inhibition tests gave the following results:

% Residual activity EDTA Pepstatin PEFABLOC PMSF Protease I 104 91 83 92Protease II 97 9 90 108

The inhibition of Protease II by Pepstatin shows that it is an asparticprotease of the Pepsin type. Protease I is not inhibited by Pepstatinand is therefore not positively identified as an aspartic protease.Optimum activity at pH 5, on the other hand, shows that it is an acidprotease. pH activity profiles are shown in FIG. 1. It is seen that bothenzymes have optimum activity at pH 5, and that Protease I is active ina more narrow range than Protease II. Thus, protease I exhibits morethan 60% activity in the range of pH 4-6, whereas Protease II exhibitsmore than 60% activity in the range of pH 4-7.

By SDS gel analysis the molecular weights of Protease I and II,respectively, were estimated to 23.000 and 37.000 kDa, respectively,From the IEF analysis of the pI of both enzymes are estimated to about4.

FIG. 2 shows the temperature-activity profiles. They are rather similarfor the two enzymes, but with slightly different optimum temperatures,50° C. for Protease I and 45° C. for Protease II.

FIGS. 3 and 4 show the pH-stability. For each pH, the zero-timeactivities have been set to 100%, and the absolute values obtained aretherefore different. Both proteases are stable at pH 4 and 5. At pH 6,Protease I is unstable, while Protease II has a certain stability (40%residual activity after 30 min). Both enzymes are unstable at pH 7.

FIGS. 5 and 6 show the temperature-stability. For each temperature, thezero-time activities have been set to 100%, and the absolute valuesobtained are different. Both proteases are stable up to 50° C., butunstable at 60° C.

Based on the results obtained on hydrolysis of insulin and glucagon byProtease I and Protease II it was found that Protease II does not reactwith insulin, whereas both proteases hydrolyse glucagon. It can beconcluded, that Protease I is a rather unspecific protease, whileProtease II is more specific. It was found that protease II is capableof cleaving the Lys-Tyr and the Phe-Val bonds found in bovine glucagon(the sequence of which is shown in Bromer, Sinn, and Behrens, J. Amer.Chem. Soc., Vol. 79, p. 2807, 1958).

EXAMPLE 4 Use of a Protease of the Invention for Viscosity Reduction

Soy flour (prepared from defatted and peeled soy beans) were pelletizedat 95° C. and grinded afterwards. The soy flour is suspended indeionized water to 15% dry substance. 5 mg protease I enzyme protein perg of dry substance and 5 mg protease II enzyme protein per g of drysubstance, respectively, was added to the soy slurry. The slurry wasincubated at 40° C. and pH 5-6. The viscosity in the slurry was measuredafter 1, 2 and 24 hours of incubation on a Brookfiled LV DV IIIviscometer using a small sample adaptor with spindle #31 at 250 rpm. Theresidual viscosities were as follows:

Prot. I Prot. II 1 hour 73% 47% 2 hours 59% 38%

EXAMPLE 5 Use of a Protease of the Invention for Cleaning of ContactLenses

In the field of contact lens cleaning it is essential to regularly haveboth an efficient disinfection and cleaning of the contact lens. One ofthe most effective ways of disinfecting contact lenses is to immersethem into a solution containing 3% H₂O₂ at pH 3.5 for at least 20minutes. The H₂O₂ is neutralized with e.g. catalase or a platinum discbefore inserting the lens into the eye. Unfortunately no commerciallyinteresting protease till date has been shown to have good effect underthese harsh conditions, so a cumbersome second step with addition of aprotease after H₂O₂-neutralization is needed to remove the proteindeposits on the contact lens. Porcine pepsin is superior to thepresently used serin protease. (Subtilisin carlsberg) but is troublesomebecause of the viruses often associated with mammal products.

Protease I and II, respectively, of the invention have been tested withrespect to the ability to remove denaturated protein from a contactlens. They have been compared to the presently used serin protease andalso to porcine pepsin, although the latter is interesting from atechnical perspective rather than a commercial perspective.

The experimental protocol was as follows:

Materials: Hen Lysozyme, L-6878 from Sigma “Rythmic” contact lens' fromEssilor (Type II lens, high-water, nonionic) Protease I produced asdescribed above Protease II produced as described above Porcine pepsin,P-6887 from Sigma Subtilisin carlsberg, Clear-Lens Pro® (Novo NordiskA/S).

Standard buffer: 0.05 M Na₂HPO₄, 0.9% NaCl pH 7.5

Reagent buffer: 0.05 M Na₂HPO₄, 0.9% NaCl, 3% H₂O₂, pH 3.5

Scintillation liquid, Optiphase “HiSafe III”

Hen lysozyme from Sigma was labelled with ¹⁴C through reductivemethylation and purified.

A solution was made containing 0.05 M Na₂HPO₄, 0.9% NaCl and 0.2 mg/mllysozyme pH 7.5. An amount of ¹⁴C-labelled lysozyme was added so the CPM(Counts Per Minute) is approximately 200.000. 1.0 ml of the solution wastransferred to a scintillation glass. The contact lens was added and theglass placed in a water-bath at 85° C. for 30 minutes.

The contact lens was then rinsed in 3×3 ml reagent buffer. It wasquartered with a scalpel. Each quarter was transferred to a newscintillation glass containing 3 ml of the reagent buffer.

Different amounts of the protease to be tested were added so the finalconcentrations were 0.1, 0.5, 2.5, 5, 12.5, 50 and 200 μg enzymeprotein/ml reagent buffer.

The reaction took place over four hours at 25° C. The quarter lenseswere rinsed in 2×3 ml standard buffer. 12 ml scintillation liquid wasadded and CPM was measured in a Packard 2500 TR liquid scintillationcounter.

Four lenses were needed to evaluate each protease: Double determinationswere made over two days, and a blind reference was needed for each lens.

The relative amount of lysozyme removed from the lens during thecombined disinfection/cleaning was calculated from the mass balance ofeach quarter lens. FIG. 7 gives a graphic presentation of theperformance of the different proteases.

Both proteases are high suitably for contact lens cleaning purposes.Protease I was found to be very superior to the other proteases.Protease II is very close to porcine pepsin in performance whereas thepresently used serin protease shows poor performance. A furtheradvantage of the acidic proteases of the invention is the low activityat the neutral pH found in tear fluid. This lowers the risk ofirritation if the lenses are not rinsed properly afterdisinfection/cleaning.

EXAMPLE 6 Use of a Protease of the Invention for Baking

Procedure:

To 10 grams of cake flour 5.9 g of water are added. The water containsdifferent concentrations of Protease I, Protease II and Neutrase®(available from Novo Nordisk A/S). The dough is mixed on a Glutamic 2200mixer for one minut. It is then placed in a plastic bag and is incubatedfor 25 min. at 32° C.

Thereafter the dough is washed with 2% NaCl (aqeuous solution) using theGlutamic mixer in order to remove starch and leave gluten in the dough.

The gluten lump is then rolled by hand untill homogeneity and is pressedinto the shape of a cylinder which is about 0.5 cm high and 2.5 cm indiameter and has a hole in the center. The gluten lump is pressed intoshape for 30 min at 25° C. Subsequently, the gluten cylinder is hung ona hook and a 2 g weight is placed in the hole. Everything is placedunder water at 25° C.

The stretching of the gluten cylinder is thereafter measured every 15min untill it breaks.

Enzymes are dosed on an Anson Unit basis (AU), initially trying with 7.5mAU/kg flour, which is the optimal dose for Neutrase®. (In theAnson-Hemoglobin method for the determination of porteolytic activitydenatured hemoglobin is digested at a temperature of 25° C., pH 7.5 anda rection time of 10 min. The undigested hemoglobin is precipitated withtrichloroacetic acid (TCA) and the amount of TCA soluble product isdetermined with phenol reagent, which gives a blue colour with tyrosineand tryptophan. 1 AU is the amount of enzyme which digests hemoglobin atan initial rate such that there is liberated per minute an amount of TCAsoluble product which gives the same colour with phenol reagent as onemilliequivalent of tyrosine).

FIG. 8 shows the stretching curves of gluten without enzyme and withNeutrase®, Protease I and Protease II. The curves are means of 6-7determinations with the same dose of enzyme. As can be seen, theaddition of all 3 proteases led to a faster stretching of the gluten.

Protease I in a dose of 7.5 mAU/kg flour does not weaken the gluten asmuch as the same dose of Neutrase®. The gluten cylinder gets longerbefore it breaks, and the rate of elongation is lower.

Addition of 2.3 mAU/kg flour of protease II, which was the largestamount possible in this system, almost weakens the gluten as much as 7.5mAU/kg of Neutrase®. The gluten breaks a little later, and the shape ofthe curves are not identical. This shows that protease II isapproximately 3 times as efficient on an AU basis as Neutrase® forgluten weakening.

In conclusion the proteases of the invention constitutes a desirablealternative to chemicals conventionally used for gluten weakening, awidely used example of which is SMS (sodium metabisulphite).

EXAMPLE 7 Use of a Protease of the Invention for Animal Feed

Ground defatted feed quality soy was mixed with deionised water underthe conditions described below. The hydrolysis was carried out in twosteps in order to simulate the pH conditions in the stomach and thesmall intestine. The performance of Protease II of the invention wascompared with that of Bio-Feed Pro, which by Brenes et al., 1993, hasbeen demonstrated to result in improved weight gain and feed efficiencywhen used in broiler diets.

Hydrolysis conditions: Hydrolysis mixture 70 g ground defatted soy 330 gdeionised water Temperature 40° C. pH 1st step 4.0 2nd step 6.5 Time 1ststep 180 minutes 2nd step 180 minutes Enzymes 1st step I) Pepsin 1.92 g(Merck art. 7190) II) I + Bio-Feed Pro 3.0 L 5.5 AU/kg soya III) I +protease II 0.19 AU/kg soya 2nd step I) Pancreatin 6 g (Sigma P 1750)II) I + Bio-Feed Pro 3.0 L 5.5 AU/kg soya III) I + Protease II 0.19AU/kg soya

Bio-Feed Pro® is available from Novo Nordisk A/S. Protease II wasobtained as described above.

The enzymes were added at start 0 minutes. During the hydrolysis °Brixand osmolality were measured to follow the reaction course. According toAdler-Nissen (1986) the osmolality values can be used for calculation ofthe Degree of Hydrolysis (DH) by the following equation:${DH} = {\frac{\Delta \quad C}{S\quad \% \times f_{osm}} \times \frac{1}{\omega} \times \frac{1}{h_{tot}} \times 100\quad \%}$

Where Δ is the increase in osmolality mOSM, S% is protein concentration,ω is the calibration factor for the osmometer, h_(tot) is the totalnumber of peptide bonds in the protein substrate (megv/g protein), andƒ_(osm) is the factor for converting % to g/kg H₂O.$f_{osm} = {\frac{1000}{100 - {{DM}\quad \%}} =}$

Further the average Molecular Weight was analysed of the sulphosalicylic acid soluble phase of the N-components of the hydrolysismixture after 90 minutes (1step) and 0, 15 and 180 minutes (2nd step).The Molecular Weight analyses were performed by the following method:

1. Principle

The sample is diluted, filtrated and injected into a liquidchromatographic system, operating in the Gel Permeation Chromatography(GPC) mode. This separation technique utilizes a liquid flow through acolumn filled with porous particles having pores with a well-definedpore diameter. When a solution of peptides having different molecularsize passes through the column, the small peptides will be able to flowinto the pores while the larger peptides will be excluded from thepores. Thus, the peptides in a solution will be separated according tomolecular size (and weight) as the large peptides will be eluted fasterfrom the column than the small peptides. A detector at the column outletcontinuously measures the effluent. The chromatographic system iscalibrated with peptides with known molecular weight.

2. Chromatographic equipment

2.1 HPLC system consisting of

High Pressure pump, WATERS M 510, flow rate 0.7 ml/min.

Injector, Waters WISP M 710

Detector, Waters M 440, with wavelength extension to 214 nm.

2.2 GPC column, 3×TSK G 2000 SWXL, 7.8 mm×300 mm, connected in seriesand operated at ambient temperature.

2.3 Integration/data processing, Waters 820 MAXIMA SIM chromatographydata system with 810/820 GPC option.

3. Reagents

3.1 Phosphate buffer, NaH₂PO₄ 2H₂O

3.2 Ammoniumchloride, NH₄CL

3.3 Trifluoroacetic acid (TFA), CF₃COOH

3.4 Acetonitrile, CH₃CN

3.5 Mobile phase:

0.05 M Phosphate buffer/0.5 M Ammoniumchloride solution containing 0.1%TFA and 25% Acetonitrile

4. Description

4.1 Calibration

The chromatographic system is calibrated by means of injections ofnumerous peptide standards with known molecular weight. The molecularweight of each standard is plotted semilogarithmic versus the observedvolume of mobile phase needed to elute the peptide from the column. By aleast squares calculation the best fitting 3rd order polynomium iscalculated. This curve represents the calibration curve.

4.2 Analysis

The sample is diluted/dissolved in mobile phase to approx. 5 mg/ml. Thesolution is filtered through a 22 μm filter and 20 μl is used forinjection into the chromatograph. The detector response versus elutionvolume is recorded. The recorded curve—the chromatogram—shows the actualmolecular weight distribution of the sample. To allow for calculationsas to accumulated weight distribution and average molecular weightcalculations, the chromatogram is divided into small time (and elutionvolume) segments—each segment being characterized by the elution volumeand the area of the chromatogram over the time interval.

5. Calculation

Results are given in terms of weight and number average molecularweights.${{{\overset{\_}{M}}_{w} = \frac{\Sigma_{i}\left( {A_{i} \star M_{w,i}} \right)}{\Sigma_{i}A_{i}}},\quad {{\overset{\_}{M}}_{n} = \frac{\Sigma_{i}A_{i}}{\Sigma_{i}\left( {A_{i}/M_{w,i}} \right)}},}\quad$

where

{overscore (M)}_(w): Weight average molecular weight

{overscore (M)}_(n): Number average molecular weight

A_(i): Area of chromatogram for each segment, measured as theaccumulated detector response over each time interval.

M_(w,i): The corresponding molecular weight for each segment. The valueis calculated by means of the calibration curve using the averageelution volume over the time interval.

RESULTS

The values for °Brix, mOSM and %DH are given in the table below:

HYDROLYSIS OF FEED SOY WITH PEPSIN, BIO-FEED PRO, PROTEASE II ANDPANCREATIN PARAMETER: ° BRIX STEP I, MIN. ENZYME 0 5 10 15 30 45 60 90120 180 PEPSIN 5.09 6.135 6.505 6.625 6.9275 7.345 7.745 8.175 8.695 9.195 BIO-FEED 5.09 6.125 6.325 6.545 7.245 7.465 7.565 8.185 8.545 9.105 PRO PROTEASE II 5.09 6.505 6.845 7.105 7.825 8.405 8.605 9.3059.705 10.205 STEP II, MIN. ENZYME 180 185 190 195 210 225 240 270 300360 PEPSIN 12.965 15.135 15.4025 15.637 16.135 16.415 16.705 17.07517.437 18.075 BIO-FEED 12.965 15.165 15.165  15.125 16.105 16.4  16.52516.885 17.285 17.805 PRO PROTEASE II 13.605 15.605 15.925  16.025 16.36516.685 16.885 17.105 17.505 18.145 PARAMETER: mOSM STEP I, MIN. ENZYME 05 10 15 30 45 60 90 120 180 PEPSIN 347 362 362 360.5 363.5 366 368 370.5373 381.5 BIO-FEED PRO 347 365 364 366 367 371 373 379 380 385 PROTEASEII 347 371 382 391 411 421 427 439 447 463 STEP II, MIN. ENZYME 180 185190 195 210 225 240 270 300 360 PEPSIN 468  996 1009 1015.5 1037.51054.5 1068.5 1087.5 1111 1148 BIO-FEED PRO 468 1013 1017 1033 1048 10581069 1094 1113 1145 PROTEASE II 548 1118 1127 1135 1152 1161 1176 12011227 1284 PARAMETER: mOSM INCREASE STEP I, MIN. ENZYME 0 5 10 15 30 4560 90 120 180 PEPSIN 0 15 15 13.5 16.5 19 21 23.5 26 34.5 BIO-FEED PRO 018 17 19 20 24 26 32 33 38 PROTEASE II 0 24 35 44 64 74 80 92 100 116STEP II, MIN. ENZYME 180 185 190 195 210 225 240 270 300 360 PEPSIN 0528 541 547.5 569.5 586.5 600.5 619.5 643 680 BIOFEED PRO 0 545 549 565578 590 601 626 645 677 PROTEASE II 0 570 579 587 604 613 628 653 679736 PARAMETER: % DH STEP I, MIN. ENZYME 0 5 10 15 30 45 60 90 120 180PEPSIN 0 2.86 2.86 2.57 3.14 3.62 4.00 4.47 4.95 6.57 BIO-FEED PRO 03.43 3.24 3.62 3.81 4.57 4.95 6.09 6.28 7.24 PROTEASE II 0 4.57 6.668.38 12.19 14.09 15.23 17.52 19.04 22.09

The results of the molecular weight analysis is given below:

Step I Step II 90 min 0 min 15 min 180 min Pepsin 960 1070 680 520Bio-Feed Pro 880 1020 690 510 Protease II 650 630 530 480

The apparent Molecular Weight is increased when pH is adjusted to 6.5 asundigested soy protein is more soluble at pH 6.5 than at pH 4.0.

It is seen that protease II releases more protein and peptides anddegrade the proteins more than Bio-Feed Pro, it therefore concluded thatprotease II is superior to Bio-Feed Pro.

Since protease II of the invention has optimum activity in acid pHrange, this enzyme will therefore perform already in the stomach of theanimal, thus an overall improvement on feed efficiency should beachieved compared to Bio-Feed Pro when applied in feed for younganimals. The above results supports this conclusion.

REFERENCES

Aviv, H. & Leder, P. 1972. Proc. Natl. Acad. Sci. U.S.A. 69: 1408-1412.

Becker, D. M. & Guarante, L. 1991. Methods Enzymol. 194: 182-187.

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. 1979.Biochemistry 18: 5294-5299.

Gubler, U. & Hoffman, B. J. 1983. Gene 25: 263-269.

Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

Sanger, F., Nicklen, S. & Coulson, A. R. 1977. Proc. Natl. Acad. Sci.U.S.A. 74: 5463-5467.

Brenes, A. et al., Poultry Science, 72, 2281-2293, 1993.

Adler-Nissen, J. Enzymic Hydrolysis of Food Proteins, Elsevier AppliedScience Publishers London and New York, 1986.

Takahashi K. et al., 1991, The Primary Structure of Aspergillus nigerAcid Proteinase A*, The Journal of Biol. Chemistry, Vol. 266, No. 29,pp. 19480-19483.

Choi, G. H. et al., Molecular analysis and overexpression of the gneeencoding endothiapepsin, an aspartic protease from Cryphonectriaparasitica, 1993, Gene 125:135-131.

Gomi, K. et al., 1993, Cloning and Nucleotide Sequence of the AcidProtease-encoding Gene (pepA) from Aspergillus oryzae, Biosci. Biotech.Biochem., 57(7):1095-1100.

Inoue, H. et al., 1991, The Gene and Deduced Protein Sequences of theZymogen of Aspergillus niger Acid Proteinase A*, The Journal ofBiological Chemistry, Vol. 266, No. 29, pp. 19484-19489.

Berka, R. M. et al., 1993, Isolation and characterization of theAspergillus oryzae gene encoding aspergillopepsin O, Gene, 125:195-198.

Berka, R. M. et al., 1990, Molecular cloning and deletion of the geneencoding aspergillopepsin A from Aspergillus awamori, Gene 86:153-162.

Berka, R. M. et al., 1990, Corrigendum, Molecular cloning and deletionof the gene encoding aspergillopepsin A from Aspergillus awamori, Gene96:313.

34 1124 base pairs nucleic acid single linear cDNA NO NO Aspergillusaculeatus 1 AATTAAGCAT CCTCCATCTT CAAAGCTCAA TCTCGCTAAC TCCCGCTCTTCTCTCGATCT 60 CATCATCCCA ATAACTCGGA CACAATGAAG ACCTCTGCTC TCTTGACCGCTGGCCTGTTG 120 GCCACCGCTG CTATTGCTGC TCCTCTCACC GAGAAGCGCG CAGCTGCTCGCGCTGCCAAG 180 CGTGGCACCA GCCGCAAGAG CAACCCCCCT CTCAAGCCCG GCACCAGCGAGGCCATCAAC 240 CTGACCGGCT CCAAGAACAC CGAGTACTCG TCCAACTGGG CCGGCGCCGTGCTCATCGGC 300 ACCGGCTACA CTGCCGTCAC CGCCGAGTTC ACCATTCCCA CCCCCTCTCTCCCCTCCGGT 360 GCCTCCAGCC GCGAGCAGTA CTGTGCCTCC GCCTGGGTCG GTATCGACGGTGACACCTGC 420 GACACCGCCA TCCTGCAGAC CGGTCTCGAC TTCTGTATCG AGGGCAGCACCGTTAGCTAC 480 GACGCCTGGT ACGAGTGGTA CCCCGACTAT GCCTACGACT TCAGCGGCATCAGCTTCTCC 540 GCCGGCGACG TTGTCAAGGT CACCGTCGAC GCCACCAGCA AGACCGCCGGTACCGCCACC 600 GTCGAGAACG TCACCAAGGG CACCACCGTC ACCCACACCT TCAGCGGTGGTGTTGATGGT 660 GATCTCTGCG AGTACAACGC CGAGTGGATC GTCGAGGACT TCGAGGAGAACTCCTCCCTC 720 GTCCCCTTCG CCGACTTCGG CACCGTCACC TTCTCCAGCG CCTACGCCACCAAGAGCGGC 780 TCCACCGTTG GTCCCTCCGG CGCCACCATC ATCGACATCG AGCAGAACAACAAGGTTCTC 840 ACCTCCGTCT CGACCTCCAG CAGCTCCGTC ACCGTCGAGT ATGTTTGAAGGGGACTCCTG 900 GGGATGTGAA GCGAGAATGC GGCTTGGGTG GTTGGAGGTC CTTTGGGACGTCGAACGCCT 960 AGGATTCAAC GGGATGAGAT CATTGGAAAT GAAGACGAGA ATGAGCGAATACTGTCACTG 1020 ATTGAGATTG TGCTTTGTTG ATTGTGTTAG GGGCTTGCCT CTGAAAATTGAGCTTAGTGT 1080 TGCTGCAATA TGATTGCTGT GGTTGAGAAA AAAAAAAAAA AAAA 11241425 base pairs nucleic acid single linear cDNA NO NO Aspergillusaculeatus 2 CTGCTTCTCC TTCTCTTCCT CCTCGTGATA TCTGCTTGAA CATCTCCTCATCATGGTCGT 60 CCTCAACAAG GCTGCAGCCC TTCTTCTGGG TCTGACCACC GCCGCCACTGCGGCTCCCCT 120 GGCCGAGAAG CAGGCTTCTG TCCCGGTCAA GAACTTCTCC GTCAAGCAGGTCGAGAAGGA 180 GGGCAGCAAG GGACGTACCG TTAACCTGCC GGGTCTGTAT GCGAATGCGCTGGCCAAGTA 240 TGGCGCCCAG GTGCCGGCCA GCGTCAAGGC CGCCGCCGTC AGTGGCAGCGTCGTGACCAC 300 CCCGCAGGCC AACGACGTCT CCTACCTGAC CCCCGTCACC GTGGGCAGCTCGACCTTGAA 360 CCTGGACTTC GACACCGGAT CCGCCGATCT CTGGGTCTTC TCCTCGGAGCTGGCCGCCTC 420 CTCGCGCACC GGCCACAGCA TCTACACCCC CGGCAGCACC GCCCAGAAGCTGTCCGGCTA 480 CAGCTGGAGC ATCTCCTACG GCGACGGCAG CTCCGCCAGC GGCGACGTCTACAAGGACAA 540 GGTCACCGTC GGCACGGTGA CGGCCAGCAG CCAGGCCGTC GAGGCCGCCAGCCGCATCAG 600 CTCCGAGTTC GTCCAGGACA CCGACACCGA CGGTCTGTTG GGTCTGGCCTTCAGCTCGAT 660 CAACACGGTC TCCCCCCGGG CCCAGACCAC CTTCTTCGAC ACCGTCAAGTCCAGCCTGGA 720 CAGCCCCCTC TTCGCCGTCG ACCTGAAGTA CCACGCCGCC GGTACCTACGATTTCGGGTT 780 CATCGACTCC TCCAAGTACA CCGGCTCCCT GACCTACGCC AACGTCGACGACTCCCAGGG 840 CTTCTGGCAA TTCACCGCCA GCGGCTACAG CGTGGGCTCG GCCTCCCACTCCTCCTCTTT 900 CTCCGCCATT GATGACACCG GCACCACCCT CATCCTCCTC GACGACTCCATCGTCTCCAC 960 CTACTACAAG AGCGTCAGCG GCGCCTCCTA CAGCTACAAC TACGGCGGCTACGTCTTCTC 1020 CTGCTCCGCC AGCCTGTCCA ACTTCAGCGT CAAGATCGGC TCCTACACCGCCGTCGTCCC 1080 CGGCAAGTAC ATCAACTACG CCCCCATCTC CACCGGCAGC TCCACCTGCTACGGCGGCAT 1140 CCAGTCCAAC GAGGGCCTCG GTCTGTCCAT CCTGGGTGAT GTCTTCCTCAAGAGCCAGCA 1200 CGTGGTCTTT GACTCGCAGG GTCCGAGAAT CGGGTTCGCC GCGCAGGCCTAGATCGTTTG 1260 ATTGGGGTTG TGGATGTGGG TGATGCTTGG TGGTGGTCTG AGTCGTGGTCTATGTGGGCG 1320 TGAATATAGT ACTGTATATA GTACTGTACA TAGGGGGGTG GTGAACATATGGTCTGGTCG 1380 ATGAATATAT GTCTTTGATG TTATGCTTCT GTGGAAAAAA AAAAA 142520 base pairs nucleic acid single linear cDNA NO NO Aspergillusaculeatus 3 AATTAAGCAT CCTCCATCTT 20 20 base pairs nucleic acid singlelinear cDNA unknown 4 CAAAGCTCAA TCTCGCTAAC 20 20 base pairs nucleicacid single linear cDNA unknown 5 TCCCGCTCTT CTCTCGATCT 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 6 CATCATCCCAATAACTCGGA 20 20 base pairs nucleic acid single linear cDNA unknown 7CAAAATGAAG ACCTCTGCTC 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 8 TCTTGACCGC TGGCCTGTTG 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 9 GCACCGCTGCTATTGCTGCT 20 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 10 CCTCTCACCG CGAAGCGCGC 20 20 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 11 ACGTGCTCGCGCTGCCAAGC 20 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 12 TGGCACCAGC CGCAAGAGCA 20 20 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 13 AGGGGGGTCTCAAGCCCGGC 20 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 14 ACCCAGCGAG GCCATAACCT 20 20 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 15 GACCGGCTCCAAGAACACCG 20 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 16 GAGGTACTCG TCCAACTGGG 20 15 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 17 CCGGCGCCGT GCCAT15 295 base pairs nucleic acid single linear cDNA NO NO Aspergillusaculeatus 18 AATTAAGCAT CCTCCATCTT CAAAGCTCAA TCTCGCTAAC TCCCGCTCTTCTCTCGATCT 60 CATCATCCCA ATAACTCGGA CAAAATGAAG ACCTCTGCTC TCTTGACCGCTGGCCTGTTG 120 GCACCGCTGC TATTGCTGCT CCTCTCACCG CGAAGCGCGC ACGTGCTCGCGCTGCCAAGC 180 TGGCACCAGC CGCAAGAGCA AGGGGGGTCT CAAGCCCGGC ACCCAGCGAGGCCATAACCT 240 GACCGGCTCC AAGAACACCG GAGGTACTCG TCCAACTGGG CCGGCGCCGTGCCAT 295 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 19 CTGCTTCTCC TTCTCTTCCT 20 20 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 20 CCTCGTGATATCTGCTTGAA 20 20 base pairs nucleic acid single linear cDNA NO NOAspergillus aculeatus 21 CATCTCCTCA TCATGGTCGT 20 20 base pairs nucleicacid single linear cDNA NO NO Aspergillus aculeatus 22 CCTCAACAAGGTGCAGCCTT 20 20 base pairs nucleic acid single linear cDNA unknown 23CTTCTGGGTC TGACCACCGC 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 24 CGCCACTGGT CCCCTGGCCG 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 25AGCCGCAGGC TTCTGTCCGG 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 26 TCAAGAACTT CTCCGTCAAG 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 27CAGGTCGAGA AGGCGGGCAG 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 28 CAAGGGACGT ACCGTTAACC 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 29TGCCGGGTCT GTATGCGAAT 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 30 GCGCTGGCCA AGTATGGCGC 20 20 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 31CCAGGTGCGG CCAGCGTCAA 20 20 base pairs nucleic acid single linear cDNANO NO Aspergillus aculeatus 32 GGCCGCCGCC GTCAGTGGCA 20 29 base pairsnucleic acid single linear cDNA NO NO Aspergillus aculeatus 33GCGTCGTGAC CACCCGCAGG CCAACGACG 29 309 base pairs nucleic acid singlelinear cDNA NO NO Aspergillus aculeatus 34 CTGCTTCTCC TTCTCTTCCTCCTCGTGATA TCTGCTTGAA CATCTCCTCA TCATGGTCGT 60 CCTCAACAAG GTGCAGCCTTCTTCTGGGTC TGACCACCGC CGCCACTGGT CCCCTGGCCG 120 AGCCGCAGGC TTCTGTCCGGTCAAGAACTT CTCCGTCAAG CAGGTCGAGA AGGCGGGCAG 180 CAAGGGACGT ACCGTTAACCTGCCGGGTCT GTATGCGAAT GCGCTGGCCA AGTATGGCGC 240 CCAGGTGCGG CCAGCGTCAAGGCCGCCGCC GTCAGTGGCA GCGTCGTGAC CACCCGCAGG 300 CCAACGACG 309

What is claimed is:
 1. A method for cleaning a contact lens comprisingtreating said contact lens with an effective amount of an isolated andpurified enzyme exhibiting protease activity at a pH of 4-7 whichexhibits protease activity in 5% hydrogen peroxide and which is encodedby a DNA sequence which hybridizes to a DNA sequence depicted in SEQ IDNO. 1 or 2 under the following conditions: hybridizing in 5×SSC,5×Denhardt's solution, 0.5% SDS and 100 ug/ml salmon sperm DNA for 16hrs. at about 65° C., followed by washes in 5×SSC, 2×SSC, 0.5% SDS,0.2×SSC, 0.5% SDS and 5×SSC at 65° C.
 2. The method according to claim1, in which said enzyme is obtainable from a filamentous fungi.
 3. Themethod according to claim 1, in which said enzyme is obtainable from astrain of Aspergillus, Rhizopus, Trichoderma, Penicillium, Fusarium,Schytalidium or Humicola.
 4. The method according to claim 1, which isobtainable from a strain of Aspergillus aculeatus.